An effective aflatoxin B1 reduction in wheat grain contaminated by Aspergillus flavus via combining the biological degradation of the toxin with inhibition of its biosynthesis

Decontamination of forage grain polluted with mycotoxins is one of the relevant problems of the forage safety provision. In recent years, the frequency of a severe contamination of forage grain and other fodder with aflatoxin B1 (AFB1) in Russia significantly increased. The probability of the AFB1 contamination of the grass stand and forage grain produced in the central and northern regions of Russia may increase in the future due to the further expansion of Aspergillus flavus fungus, the main AFB1 producer, into these regions as a result of climate change. One of the promising approaches to decontaminate grain contaminated with AFB1 is the toxin catabolization by various microorganisms producing enzymes able to degrade AFB1. Another approach includes the treatment of grain contaminated with the AFB1 producers with compounds able to inhibit the aflatoxigenesis. In the present work, it was shown for the first time that the treatment of the cultural broth of Rhodococcus erythropolus AC-884 with the supernatant after the treatment with compactin almost completely prevents the accumulation of mycotoxin in the infected grain. The aim of the work is to evaluate the effectiveness of reducing the content of mycotoxin in wheat grain artificially contaminated with aflatoxin B1 after treatment with actinobacteria of the genus Rhodococcus or an inhibitor of aflatoxygenesis, the compactin, as well as a combination of these methods. The present study reports the results of investigation of the AFB1 destruction capability in four Rhodococcus strains ( Rhodococcus sp., AC-1260, R. erythropolus AC-1269 and АС-884, and R. ruber AC-1801). Quantitative analysis of AFB1 by high performance liquid chromatography revealed that the most active mycotoxin degradation occurred in the cell-free cultural broth supernatant of АС-884 (СBS-884). Only trace amounts of AFB1 added in CBS-17 to a final concentration of 0.2 µg/ml were detected in this supernatant after 48-h incubation at 30 °C, whereas cultural broth supernatants of other studied strains contained from 15 to 50 % of the added AFB1 after its incubation under the same conditions. A 72-h treatment of wheat ( Triticum aestivum L., cv. Daria) grain artificially contaminated with AFB1 (1.0, 2.5, or 5.0 mg/g) with CBS-884 removed 60 % of the toxin, while the use of cultural broth supernatants or cell suspensions of AC-1260, AC-1269 or AC-1801 strains did not result in any changes in the AFB1 content comparing to the control. Using a consecutive treatment of grain infected with a toxigenic Aspergillus flavus strain by compactin, inhibiting the AFB1 production in this fungus, and then by CBS-884, we first demonstrated that the approach based on application of AFB1 biosynthesis inhibitors followed by the toxin biodegradation allowed an efficient decontamination of grain if the use of inhibitors alone did not result in a complete suppression of the aflatoxigenesis. Grain treatment of with the supernatant of AC-884 was more effective than the treatment with a similarly obtained supernatant of another АFB1-destroiyng agent, Phoma glomerata PG-41. In our experiments, the AFB1 content in wheat grain contaminated with A. flavus А11 reduced twice compared to the control in 7 days after compactin (0.05 mg/g) application. In 24 h after the treatment of the contaminated grain with CBS-884 alone (0.25 ml/g), the AFB1 amount produced by A. flavus for six post-inoculation days was reduced almost thrice. Combination of both treatments (compactin, 0.05 mg/g, and then CBS-884, 0.25 ml/g) resulted in a more than 200-fold reduction of the AFB1 content and the achievement of almost complete grain decontamination. Therefore, an approach based on a combination of biodegradation with inhibition of aflatoxigenesis can provide effective decontamination of grain contaminated with AFB1 producers in cases where the use of inhibitors does not lead to complete suppression of mycotoxin biosynthesis.